Green Fluorescent Protein (GFP) from the hydromedusa Aequorea victoria (synonym A. A.), described by Johnson et al. in J Cell Comp Physiol. (1962), 60:85-104, was found as a part of bioluminescent system of the jellyfish where GFP played the role of a secondary emitter transforming blue light from the photoprotein aequorin into green light.
cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene, 1992, V. 111 (2), pp. 229-233). It turned out that this gene can be heterologically expressed in practically any organism due to unique ability of GFP to form a fluorophore by itself (Chalfie et al., Gene (1992), 111(2):229-233). This finding opens broad perspectives for use of GFP in cell biology as a genetically encoded fluorescent label.
A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful and optimized for a variety of research purposes. New versions of GFP have been developed, such as a “humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells (Haas, et al., Current Biology 1996, V. 6, pp. 315-324; Yang, et al., Nucleic Acids Research 1996, V. 24, pp. 4592-4593). One such humanized protein is “enhanced green fluorescent protein” (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions. Also, GFP variants with improved folding and cellular fluorescence under incubation at 37° C. have been obtained. Useful A. victoria GFP mutants are described in detail in U.S. Pat. Nos. 5,491,084, 5,625,048, 5,777,079, 5,804,387, 6,090,919, 5,874,304, 5,968,750, 6,020,192, 6,027,881, 6,046,925, 6,054,321, 6,066,476, 6,096,865, 6,146,826, 6,414,119, 6,638,732, 6,699,687, 6,803,188, 6,077,707, 6,124,128, 6,172,188, 6,818,443, 6,194,548, 6,265,548, 6,319,669, 6,403,374, 6,593,135, 6,800,733, 6,780,975, 6,852,849, and 6,919,186.
GFP homologs from different species including Anthozoa and Arthropoda were isolated (Matz et al., Nature Biotechnol. 1999, V. 17, pp. 969-973; Shagin et al., Mol Biol Evol. 2004, V. 21(5), pp. 841-850). A number of biological and biomedical applications of these proteins are discussed in detail by Lippincott-Schwartz and Patterson in Science, 2003, V. 300(5616), pp. 87-91. Also, close homologues of A. victoria GFP were isolated from other jellyfishes the of Aequorea genus including A. macrodactyla green fluorescent protein, GFPxm (Xia et al., Mar Biotechnol 2002, V. 4(2), pp. 155-62) and A. coerulescens GFP-like protein, AcGFPL (Gurskaya et al., Biochem J. (2003), 373(Pt 2): 403-408).
A. macrodactyla GFPxm shares 83% identity with A. victoria GFP. Wild type GFPxm is not useful as a fluorescent marker in cell-based assays because of a low maturation speed at 37° C. Modification of GFPxm to optimize its maturation speed at temperatures of 35-39° C. provide a means for detecting the reporter in mammalian cells at lower levels of expression and/or increased sensitivity relative to wild type GFPxm. This greatly improves the usefulness of the GFPxm in studying cellular functions in living cells.